Cloning LFA-1

ABSTRACT

The present invention discloses a cDNA sequence which encodes the beta-subunit of LFA-1. The present invention further discloses fragments of the cDNA sequence which are capable of recognizing a restriction length polymorphism within the LFA-1 sequence.

This application is a continuation of application Ser. No. 07/771,849,filed Oct. 7, 1991, abandoned, which is a continuation of applicationSer. No. 07/019,440, filed Feb. 26, 1987, abandoned.

BACKGROUND OF THE INVENTION

The work described herein was performed with the aid of governmentfunding, and the government therefore has certain rights in theinvention. Specifically, the work was supported by N.I.H. grants CA31798 and AI 05877.

This invention relates to cellular adhesion.

Cellular adhesion is a critical function for guiding migration andlocalization of cells, and for maintaining the integrity of the body.Receptors for extracellular matrix components such as fibronectin,laminin, and vitronectin mediate cellular adhesion during morphogenesisand wound healing. In the immune system, regulatory networks requireintimate cell-cell interaction among lymphocytes and antigen-presentingaccessory cells, and cell-mediated cytolysis involves direct contactbetween the effector cell and virally-infected or transformed targetcells. Leukocyte-endothelial interactions are important in leukocytemobilization into inflammatory sites and in lymphocyte recirculation.These cellular adhesion reactions are mediated in part by a family ofstructurally related glycoproteins, LFA-1, Mac-1, and p150,95, all ofwhich share a common β-subunit (hereinafter referred to as the β-subunitof human LFA-1). Springer et al., 314 Nature 540, 1985; Springer et al.,"The lymphocyte function-associated LFA-1, CD2, and LFA-3 molecules:cell adhesion receptors of the immune system" Ann. Rev. Immunol. Vol. 5,1987; both hereby incorporated by reference.

SUMMARY OF THE INVENTION

In general, the invention features a) substantially pure recombinantβ-subunit of a human glycoprotein concerned with cellular adhesion, orb) a biologically active fraction of this β-subunit, c) an analog of theβ-subunit, or c) a fragment of the β-subunit, composed of at least 10%of a contiguous sequence of the β-subunit. The invention also features acDNA sequence encoding for the β-subunit; and a vector containing a DNAsequence encoding therefor. By recombinant subunit is meant thepolypeptide product of recombinant DNA encoding the β-subunit, i.e., thepolypeptide expressed from DNA which is not in its naturally occurringlocation within a chromosome. By natural subunit is meant that subunitproduced naturally in vivo from naturally occurring and located DNA. Byanalog is meant a polypeptide differing from the normal polypeptide byone or more amino acids, but having substantially the biologicalactivity of the normal polypeptide. The invention also features anymonoclonal antibody (MAb) raised against the recombinant β-subunit, abiologically active fraction, an analog, or a fragment thereof composedof at least 10%, preferably at least 80%, of a contiguous sequence ofthe β-subunit of a human glycoprotein.

The cDNA sequence encoding the LFA-1 β-subunit or a fragment thereof maybe derived from any of the naturally occurring genes encoding it, orsynthesized chemically. Variations in this sequence which do not alterthe amino acid sequence of the resulting protein, or which do notsignificantly alter the biological activity of the protein, are alsoacceptable, and are within this invention.

Preferably the human glycoprotein is LFA-1, Mac-1 or p150,95.

As will be described in more detail below, the invention permits thediagnosis and treatment of a variety of human disease states.

Other features and advantages of the invention will be apparent from thefollowing description of the preferred embodiments thereof and from theclaims.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The drawings are first briefly described.

DRAWINGS

FIGS. 1A to 1F is the DNA coding sequence of the β-subunit of LFA-1,Mac-1 and p150,95. Potential N-glycosylation sites are marked withtriangles.

FIG. 2 is a comparison of the amino acid sequence predicted from thecDNA in FIGS. 1A to 1F, and the amino acid sequence derived from enzymedigests of the β-subunit of LFA-1. Ambiguous determinations of aminoacids are bracketed. The code for amino acids is as follows:

    ______________________________________                                        Ala,     A              alanine                                               Arg,     R              arginine                                              Asn,     N              asparagine                                            Asp,     D              aspartic acid                                         Cys,     C              cysteine                                              Gln,     Q              glutamine                                             Glu,     E              glutamic acid                                         Gly,     G              glycine                                               His,     H              histidine                                             Ile,     I              isoleucine                                            Leu,     L              leucine                                               Lys,     K              lysine                                                Met,     M              methionine (start)                                    Phe,     F              phenylalanine                                         Pro,     P              proline                                               Ser,     S              serine                                                Thr,     T              threonine                                             Trp,     W              tryptophan                                            Tyr,     Y              tryosine                                              Val,     V              valine                                                ______________________________________                                    

METHODS

In general, the β-subunit of any of the above described relatedglycoproteins is isolated by standard procedures and the amino acidsequence of at least a part of it determined. From this analysis asynthetic oligonucleotide probe, corresponding to the amino acidsequence, is synthesized and used as a probe for a genomic or cDNAlibrary containing a DNA sequence encoding the β-subunit. An example ofthis procedure is given below. One skilled in the art will realize thatthis represents only one of many methods which can be used to achievecloning of the gene encoding the LFA-1 β-subunit.

Purification of the β-Subunit

MAb's directed against the alpha subunits of p150,95, Mac-1, and LFA-1,were used to affinity purify their respective proteins from threedifferent sources. The p150,95 protein was purified from hairy cellleukemia spleens (Miller et al., 1986, 137 J. Immunol. 2891, herebyincorporated by reference); Mac-1 was purified from pooled humanleukocytes (Miller et al., supra); and LFA-1 was purified from the SKW3T cell line using TS1/22 monoclonal antibody (Sanchez-Madrid et al.1983, J. Exp. Med. 158:586, hereby incorporated by reference).

Preparative SDS-PAGE gels were run using the method of Laemmli(Hunkapiller et al., 1983, Meth. Enzym. 91:227). 0.1 mM Na Thioglycolatewas added to the upper chamber to reduce the level of free radicals inthe gel. Bands were visualized by soaking the gel for several minutes in1 M KCl and then excised. The β-subunit was electroeluted using theapparatus and method described by Hunkapillar et al., supra. Thepurified protein was reduced with 2 mM DTT in the presence of 2% SDS andalykylated with 5 mM iodoacetic acid in the dark. (In some cases, theprotein was reduced and alkylated prior to running the preparative gel.)

Amino Acid Sequencing

The above samples were precipitated using four volumes of ethanol at-20° C. for 16 hr, and the protein pellet redissolved in 30-50 μl of 0.1M NH₄ CO₃ containing 0.1 mM CaCl₂ and 0.1% zwittergent 3-14 (Calbiochem,San Diego, Calif.). The sample was then digested with 1% w/w trypsin for6 hr at 37° C. At 2 and 4 hr during the incubation, additional trypsin(1% w/w) was added.

The tryptic peptides were resolved by reverse phase HPLC (BeckmanInstruments) with a 0.4×15 cm C4 column (Vydac, Hesperig, Calif.), andeluted from a 2 hr linear gradient from 0 to 60% acetonitrile. 0.1% TFAwas included in both the aqueous and organic solvents. The peaks weremonitored at 214 and 280 nm and collected into 1.5 ml polypropylenetubes. The fractions were concentrated to 30 μl or less on a speed-vacapparatus, and selected peptides subjected to sequence analysis using agas phase microsequenator (Applied Biosystems, Foster City, Calif.).

EXAMPLE β-subunit of p150,95

p150,95 was affinity purified from the spleens of human patients withhairy cell leukemia using a monoclonal antibody specific for the alphasubunit (MW approx. 150,000, Miller et al., supra). Analysis of thepurified protein by SDS-PAGE and silver staining revealed thecharacteristic alpha and beta subunit, with no significant amounts ofcontaminating proteins. The β-subunit band was excised from apreparative SDS-PAGE gel and electroeluted, as described above.

The N-terminus of the beta subunit was blocked and therefore could notbe sequenced. Internal amino acid sequence information was obtained bydigesting the β-subunit with trypsin. The tryptic peptides were resolvedby reverse phase HPLC and eluted on a 60% acetonitrile gradient. Peaksanalyzed by absorbance at 214 and 280 nm were collected and applied to agas phase microsequenator.

The peptide sequences of two of these fragments is: P-61 PeptideSequence: LeuTyrGluAsnAsnIleGlnProIlePheAlaValThrSer P-20 PeptideSequence: ThrAspThrGlyTyrIleGlyLys.

Two strategies were adopted for constructing oligo-nucleotide probes. Aunique sequence 39mer was designed from peptide P-61 based on humancodon usage frequency (Lathe, 1985, J. Mol. Biol. 183:1). Its sequenceis: 3'-GACATACTCTTGTTGTAGGTCGGGTAGAAACGACACTGG -5'. In addition, twosets of mixed sequence probes were constructed such that every possiblesequence was represented. A 20mer of 96-fold redundancy was derived frompeptide P-61, and a 17 mer of 192-fold redundancy was constructed basedon the sequence from a different peptide fragment of the β-subunit,P-20. These sequences are given below. ##STR1##

The 39mer and the mixed sequence 20mer were used to probe a Northernblot of poly A+selected RNA from PMA-activated U937 cells. The U937cells, JY lymphoblastoid cells, HeLa cells, and CO3 cells (Springer etal., 1984, J. Exp. Med. 160:1901, an EBV-transformed cell line from ahealthy donor) were grown in RPMI 1640 containing 10-15% fetal calfserum in a humidified atmosphere of 5% CO₂ and 37° C. The U937 cellswere activated with 2 ng/ml PMA for three days prior to harvesting. Thecells were lysed in a 4M guanidinium isothiocyanate solution, and RNAisolated in a 5.7M CsCl gradient. Poly A+ mRNA was selected with oligo(dT)-cellulose columns (Maniatis et al., Molecular Cloning: A laboratorymanual, Cold Spring Harbor Laboratory, N.Y., 1982) or oligo(dT)-affinity paper (Amersham). This RNA was denatured and sized on a 1%agarose gel containing formaldehyde (Maniatis et al., supra) andtransferred to nylon membranes (BioRad) in 20X SSC. A lane containing28S and 18S ribosomal RNA from human cells or 23S and 16S rDNA fromEscherichia coli was run to provide molecular weight standards.

The filters were hybridized with nick-translated probe DNA at 42° C. for18 hr in 5 X SSPE, 50% formamide, 10% dextran sulfate, 1 X Denhardts,0.5% SDS and 100 ug/ml denatured salmon sperm DNA, and washed at highstringency (65° C.) in 0.2X SSC and 0.1% SDS. Both probes identified aband of approximately 3 kb. The 39mer gave a much stronger signal andwas chosen for the primary screening of a cDNA library.

A human tonsil cDNA library (gift of L. Klickstein) was size-selectedfor inserts of 2 kb or greater and constructed in λt11 (Wong et al.,1985, Proc. Nat. Acad. Sci. U.S.A. 82:7711). The original library of4×10⁶ recombinants was amplified once, and 200,000 recombinants platedat a density of 7500 plaques/100 mm plate. The plaques were amplified insitu on duplicate nitrocellulose filters, as described by Woo (1979,Meth. Enzym. 68:389).

The oligonucleotide probes were labeled with ³² P-ATP usingpolynucleotide kinase. The filters were prehybridized for at least 2 hrat 42° C. in 6 X SCC, 1 X Denhardts, 0.5% SDS, 0.05% phosphate buffer,and 100 μg/ml of salmon sperm DNA. Hybridization with the 39mer wasovernight at 42° C. in prehybridization solution containing 20 μg/mltRNA. The filters were washed at 53° C. to 55° C. with 6 X SSC, 0.1%SDS, and 0.05% phosphate buffer. The damp filters were covered withplastic wrap and exposed to film with an intensifying screen. Phage thatgave positive signals on duplicate filters were plaque purified andrescreened with the 39mer at a higher wash temperature (60° C.) and with20mer and 17mer mixed sequence probes. 15 positive clones were picked.Eight of the clones crossreacted with each other and gave positivesignals with the 20mer mixed sequence probe and the independent 17mermixed sequence probe. These clones were chosen for further analysis.

To confirm the identity of the cDNA clones, a 263 bp PstI/EcoRIrestriction fragment which hybridized to the 39 mer was subcloned intoM13 vector and sequenced by the Sanger dideoxy chain termination methodas follows. The amino acid sequence deduced from the DNA sequence isidentical in 13 of 14 positions to the peptide sequence from which the39mer probe was derived, including one amino acid which was not includedin the design of the oligonucleotide. Furthermore, the predicted aminoacid sequence shows that this peptide is preceded by a lysine andfollowed by an arginine, as expected for a tryptic fragment. The onemismatch may be due to normal polymorphism. The unique sequenceoligonucleotide was 87% homologous to the cDNA sequence, despite the oneamino acid mismatch.

The cDNA clones were restriction mapped by single and double restrictiondigests and, after end-labeling, by partial restriction digests(Maniatis et al., supra). Compatible restriction fragments weresubcloned directly into M13 cloning vectors. Other fragments were firstblunt ended with Klenow, T4 polymerase, or Mung Bean nuclease (Maniatiset al., supra) and ligated into the HincII or SmaI site of the M13polylinker. The nucleotide sequence of both strands was determined bythe dideoxy chain termination method of Sanger et al. (1977, Proc. Nat.Acad. Sci. U.S.A. 74:5463) using ³⁵ S-dATp.

The complete nucleotide sequence and deduced amino acid sequence of theβ-subunit gene in the longest clone, 18.1.1 (2.8 kb is length), is shownin FIGS. 1A to 1F. The first ATG is at position 73, and the sequencesurrounding the ATG is consistent with the consensus rules for aninitiation codon (Kozak 1984, Nucl. Acid. Res. 12:857). This putativeinitiation codon is followed by an open reading frame of 2304 bp, whichcould encode a polypeptide of 769 amino acids (aa). The stop codon ATCis followed by a 3' untranslated region of 394 bp. The poly A tail wasnot found, although a consensus polyadenylation signal (AATAAA) islocated 9 bp from the 3' end.

The deduced amino acid sequence of the cDNA clones was compared topeptide sequence data from the beta subunit of Mac-1, LFA-1, and p150,95(FIG. 2). In addition to the P61 and P-20 peptide sequences given above,one other peptide was sequenced from the beta subunit of p150,95.Tryptic peptides were also prepared and analyzed from the beta subunitof purified Mac-1 and LFA-1. Each peptide sequence is found within thededuced amino acid sequence (FIGS. 1 and 2). Thus, it can be concludedthat the cDNA encodes the β-subunit of human LFA-1.

The cDNA clones hybridize to a single mRNA species of approximately 3.0kb, which is the same message identified by the 39mer oligonucleotide.This message is present in PMA-activated U937 cells (LFA-1⁺, Mac-1⁺,p150,95⁺), JY lymphoblastoid cells (LFA-1⁺, Mac-1⁻, p150,95⁻), andEBV-transformed cells from a normal donor (LFA-1⁺, Mac-1⁻, p150,95⁻),but is absent in HeLa cells (LFA-1⁻, Mac-1⁻, p150,95⁻). Although clone18.1.1 lacks the poly A tail, it is close to the estimated size of theRNA message.

Within the deduced polypeptide are two regions of sufficient length andhydrophobicity that could span the membrane bilayer. The first domain,which begins with the putative initiation methionine and extends 22amino acids, has the characteristics of a signal sequence. This putativesignal sequence is followed by a charged glutamine, a residue which isoften cyclized at the N-terminal position. This would be consistent withthe N-terminal blockage of the β-subunit, if the signal sequence iscleaved during processing.

Use

The cDNA encoding the β-subunit of human LFA-1 can be used to producerecombinant β-subunit in large amounts. For example, thebeta-subunit-encoding cDNA can be excised from the λgt11 clones andintroduced into an expression vector (plasmid, cosmid, phage or othertype) to express the μ-subunit in E. coli, using standard techniques.Alternatively the clones may be inserted into other vectors, such asmammalian, insect, or yeast expression vectors, and used to producerecombinant β-subunit in mammalian or yeast cells.

The subunits produced by the above methods can be readily purified andused as an immunogen to raise monoclonal antibodies to the subunits.These antibodies can be labelled and used in standard immunoassays tomonitor the level of LFA-1, Mac-1, or p150,95 in white blood cells, andin the serum or other body fluids of patients having medical disordersassociated with too many or too few cells having on their surfaces LFA-1or related proteins. For example, diseases, e.g., AIDS, characterized byimmunosuppression can be expected to be accompanied by abnormally lowlevels of such cells, which are instrumental in fighting infections, andsuch diseases can thus be monitored by monitoring levels of theseproteins. Also, other disease states, e.g., autoimmune disease,allograft rejection, and graft-versus-host disease, can be expected tobe characterized by abnormally high levels of such cells, and thus alsocan be monitored by monitoring levels of these proteins. They can alsobe used to diagnose leukocyte adhesion deficiency, an inheriteddeficiency in the LFA-1, Mac-1, and p150,95 glycoproteins. Antibodies tothe β-subunit can also be used to purify LFA-1 or related proteins byconventional immunoaffinity purification methods.

The purified proteins, particularly LFA-1, Mac-1 and/or p150,95, whethernative or recombinant, can also be used therapeutically. The proteinscan be administered to patients in need of such treatment in aneffective amount (e.g., from 20-500 μg per kg body weight), and mixedwith a pharmaceutically acceptable carrier substance such as saline.Therapeutic utility of these proteins is based on the fact that diseasestates such as autoimmune diseases, allograft rejections, andgraft-versus-host diseases involve abnormally high levels ofcell-to-cell contact mediated by the recognition and binding of LFA-1and related proteins to target antigen presenting cells, endothelialcells, and other types of cells. The administration of LFA-1 or arelated protein, or fragments thereof, will compete for receptors forthe cell-bound protein, inhibiting cell-to-cell binding and thusbringing about the desired immunosuppression. A particular disease forwhich these proteins will be useful is the autoimmune disease rheumatoidarthritis. Preferably administration is intravenous at about 20-500 μgper kg body weight, or directly at an inflamed joint of a patientsuffering from rheumatoid arthritis. Alternatively, oral administrationor local application can be used by providing tablets, capsules, orsolutions, or by applying lotions as required. The amount and method ofadministration will vary dependent upon the age and weight of thepatient, and the disease to be treated. Other automimmune diseases whichcan be treated include systemic lupus erythematosis, juvenile onsetdiabetes, multiple sclerosis, allergic conditions, eczema, ulcerativecolitis, inflammatory bowel disease, Crohn's disease, as well asallograft rejections (e.g., rejection of a transplanted kidney orheart). LFA-1, Mac-1, and p150,95 noramlly act in situ by binding toendothelial and other cells. Thus, the free proteins or peptides, whichare administered, will be able to inhibit leukocyte immune responses andmigration to inflammatory sites.

The β subunit cDNA clone can be used in prenatal diagnosis of leukocyteadhesion deficiency (LAD). LAD disease is a deficiency in cell surfaceexpression of LFA-1, Mac-1, and p150,95 and is due at least in part to aprimary genetic lesion in the β subunit. Patients with the severe formof LAD disease suffer from recurrent bacterial infections and rarelysurvive beyond childhood. The defect can be detected early in pregnancysince it is associated with a unique restriction fragment lengthpolymorphism. PstI digestion of human DNA and hybridization with the 1.8kb EcoRI fragment (shown in FIG. 2) of the β subunit cDNA defines arestriction fragment length polymorphism (RFLP). Diagnosis of thisdisease is therefore performed by standard procedure using the whole ora part of this EcoRI fragment. The genomic DNAs of the parents of thefetus, and the fetus are screened with this probe and an analysis oftheir RFLPs made. In this way the probability that the fetus has thedisease can be estimated.

Other embodiments are within the following claims.

We claim:
 1. An isolated nucleic acid molecule which encodes the amino acid sequence of the common beta subunit of a human glycoprotein selected from the group consisting of LFA-1, MAC-1 and p150,95.
 2. A fragment of the isolated nucleic acid molecule of claim 1, wherein said fragment detects a restriction fragment length polymorphism in the genomic region encoding the common beta subunit of a human glycoprotein selected from the group consisting of LFA-1, MAC-1 and p150,95, and further wherein said restriction fragment length polymorphism is associated with leukocyte adhesion deficiency.
 3. The fragment of claim 2 wherein said fragment of said cDNA is the 1.8 kb EcoRI fragment.
 4. The fragment of claim 2 wherein said restriction length polymorphism is located within a PstI fragment of said genomic region.
 5. The isolated nucleic acid molecule of claim 1, wherein said isolated nucleic acid molecule consists of the nucleotide sequence depicted in FIG.
 1. 6. The isolated nucleic acid molecule of claim 1, wherein said isolated nucleic acid molecule carries a label.
 7. An isolated nucleic acid molecule having a sequence which is the reverse complement of the sequence of the isolated nucleic acid molecule of claim
 1. 8. The isolated nucleic acid molecule of claim 7, wherein said isolated nucleic acid molecule carries a label.
 9. A vector containing the isolated nucleic acid molecule of claim
 1. 10. An isolated nucleic acid molecule which encodes a polypeptide amino acid sequence of the common beta subunit of a human glycoprotein selected from the group consisting of LFA-1, MAC-1 and p150,95, wherein said said polypeptide amino acid sequence consists of the amino acid sequence depicted in FIG.
 1. 11. The isolated nucleic acid molecule of claim 10, wherein said isolated nucleic acid molecule carries a label. 